Undetected isoniazid mono resistance in rural Eastern Cape Province - A risk for the emergence of multidrug-resistant TB

Thesis (MSc)--Stellenbosch University, 2021.ENGLISH ABSTRACT: The emergence of drug-resistant tuberculosis (TB) remains a major challenge in South Africa, particularly the Eastern Cape, being one of the most severely affected provinces in the country. Due to resource limitations, many isoniazid (INH) mono-resistant TB cases remain undiagnosed, as TB control programmes generally focus on rifampicin-resistant strains. Rifampicin resistance, which is a marker of multidrug-resistant (MDR) TB, is more difficult to treat than INH mono-resistant (IMR) TB, often resulting in high morbidity and mortality. The effectiveness of INH, an important first-line anti-TB drug, has been compromised by resistance, which arises through spontaneous mutations in the genome of Mycobacterium tuberculosis (M. tuberculosis). Occurrence of these mutations is often missed by the current diagnostic algorithm, which fails to detect IMR-TB cases at diagnosis, hence threatening the efficacy of TB treatment. Consequently, these patients are likely to be treated with a weakened regimen, which may increase the risk of treatment failure or relapse. Previous studies have reported IMR as the source for the emergence of MDR-TB. This study aimed to provide the first in-depth analysis of the molecular epidemiology of IMR-TB in the Eastern Cape. Clinical isolates from patients with rifampicin-susceptible TB were obtained via the National Health Laboratory Services (NHLS) in Port Elizabeth and analysed by using a series of microbiological and molecular techniques. These tests were done to identify IMR-TB cases, describe the molecular mechanisms of INH resistance, identify cases of acquisition of IMR and MDR, as well as to describe risk factors associated with IMR at diagnosis (baseline). We also used spoligotyping to classify isolates into their respective lineages and strain families. Phenotypic INH drug susceptibility testing on solid media identified 107 (13.9%) cases of IMR among the cohort of 993 TB cases enrolled, which was nearly double the estimated national average. No association between patient demographic or clinical parameters was identified. This may be due to the inaccuracies of the electronic TB database. Genetic drug susceptibility testing only identified causal mutations in 25 baseline isolates, while 4 baseline isolates showed evidence of heteroresistance, possibly masking the detection of underlying INH-resistant populations. This was confirmed in a small sub-analysis using a highly sensitive targeted deep sequencing approach. Subsequent analysis of serial isolates showed acquisition of IMR in 9 cases, as well as loss of IMR in 12 cases. Repeat analysis identified heteroresistance as the possible cause of the observed flip flopping of the IMR phenotype. Spoligotyping failed to identify reinfection as a major mechanism causing the flip flopping IMR phenotype. IMR was associated with the Atypical Beijing genotype (p < 0.0001). This study highlights the need to change TB policy through: (1) understanding the local epidemiology of IMR to identify potential risk factors for targeted interventions and to strengthen current first-line regimens for the continuation phase of TB treatment, (2) improving surveillance studies in neglected rural areas by monitoring IMR to inform policy, (3) developing new rapid molecular technologies to ensure early identification of IMR-TB cases and close monitoring of patients following appropriate treatment and care. These strategies will be essential to contain the spread of IMR-TB, improve outcomes and prevent progression of disease to more severe forms of drug resistance often culminating in death.AFRIKAANSE OPSOMMING: Die opkoms van middelweerstandige tuberkulose (TB) bly 'n groot uitdaging in Suid-Afrika, veral die Oos-Kaap, as een van die provinsies in die land wat die ergste geraak word. As gevolg van hulpbronbeperkte kapasiteit, bly baie isoniasid (INH) mono-weerstandige TB gevalle nie gediagnoseer nie, aangesien TB-beheerprogramme oor die algemeen fokus op rifampisin-weerstandige stamme. Rifampisin weerstand is 'n merker van multimiddel weerstandige (MDR) TB, wat moeiliker is om te behandel as INH mono-weerstandige (IMR) TB, wat dikwels lei tot hoë morbiditeit en mortaliteit. Die effektiwiteit van INH, 'n belangrike eersterangse middel teen TB, word verminder deur weerstand wat ontstaan deur spontane mutasies in die genoom Mycobacterium tuberculosis (M. tuberculosis). Die voorkoms van hierdie mutasies word dikwels gemis deur die huidige diagnostiese algoritme, wat nie IMR-TB-gevalle kan opspoor tydens diagnose nie. Gevolglik word die doeltreffendheid van TB-behandeling bedreig, aangesien hierdie pasiënte meer geneig is om met 'n verswakte behandeling behandel te word, wat die risiko van versuim of terugval kan verhoog. Vorige studies het IMR as die bron vir die opkoms van MDR-TB gerapporteer. Hierdie studie het ten doel gehad om die eerste diepgaande analise van die molekulêre epidemiologie van IMR-TB in die Oos-Kaap te bied. Kliniese isolate van pasiënte met rifampisin-vatbare TB is verkry deur die National Health Laboratory Services (NHLS) in Port Elizabeth en geanaliseer deur gebruik te maak van 'n reeks mikrobiologiese en molekulêre tegnieke. Hierdie toetse is gedoen om IMR-TB-gevalle te identifiseer, die molekulêre meganismes van INH-weerstand te beskryf, gevalle van verkryging van IMR en MDR te identifiseer, asook om risikofaktore wat verband hou met IMR tydens diagnose (basislyn) te beskryf. Ons het ook spoligotipering gebruik om isolate in hul onderskeie geslagte en stamfamilies te klassifiseer. Fenotipiese toetsing vir vatbaarheid vir INH-geneesmiddels op vaste media het 107 (13,9%) gevalle van IMR geïdentifiseer onder die groep 993 TB-gevalle wat ingeskryf is, wat byna dubbel die geskatte nasionale gemiddelde was. Geen verband tussen pasiënte se demografiese of kliniese parameters is geïdentifiseer nie. Dit kan te wyte wees aan die onakkuraathede van die elektroniese databasis. Die toetsing van vatbaarheid vir genetiese geneesmiddels het slegs oorsaaklike mutasies in 25 basislyn-isolate geïdentifiseer, terwyl vier basislyn-isolate bewyse van heteroresistensie getoon het, wat moontlik die opsporing van onderliggende INH weerstandige populasies kon wegsteek. Dit is bevestig in 'n klein sub-analise deur gebruik te maak van 'n baie sensitiewe, gerigte diepvolgorde-benadering. Daaropvolgende ontleding van reeksisolate het die verkryging van IMR in 9 gevalle getoon, asook verlies aan IMR in 12 gevalle. Heranalise het heteroresistensie geïdentifiseer as die moontlike oorsaak van die waargenome “flip-flopping” van die IMR-fenotipe. Spoligotipering kon nie herinfeksie identifiseer as 'n belangrike meganisme wat die IMR-fenotipe omkeer. IMR word geassosieer met die Atipiese Beijing-genotipe (p